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1.
Two-dimensional electrophoresis map of the human hepatocellular carcinoma cell line, HCC-M, and identification of the separated proteins by mass spectrometry 总被引:32,自引:0,他引:32
Currently, one of the most popular applications of proteomics is in the area of cancer research. In Africa, Southeast Asia, and China, hepatocellular carcinoma is one of the most common cancers, occurring as one of the top five cancers in frequency. This project was initiated with the purpose of separating and identifying the proteins of a human hepatocellular carcinoma cell line, HCC-M. After two-dimensional gel electrophoresis separation, silver staining, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analyses, tryptic peptide masses were searched for matches in the SWISS-PROT and NCBI nonredundant databases. Approximately 400 spots were analyzed using this approach. Among the proteins identified were housekeeping proteins such as alcohol dehydrogenase, alpha-enolase, asparagine synthetase, isocitrate dehydrogenase, and glucose-6-phosphate 1-dehydrogenase. In addition, we also identified proteins with expression patterns that have been postulated to be related to the process of carcinogenesis. These include 14-3-3 protein, annexin, prohibitin, and thioredoxin peroxidase. This study of the HCC-M proteome, coupled with similar proteome analyses of normal liver tissues, tumors, and other hepatocellular carcinoma cell lines, represents the first step towards the establishment of protein databases, which are valuable resources in studies on the differential protein expressions of human hepatocellular carcinoma. 相似文献
2.
Chi-Yu Lee Andrew Leigh-Brown Charles H. Langley Daniel Charles 《Applied biochemistry and biotechnology》1977,2(3):213-224
Recently in our laboratory, up to 20 different enzymes and their genetic variants have been purified from mouse andDrosophila by affinity chromatography. By virtue of the specific coenzyme requirements, up to ten different enzymes could be copurified from a single tissue extract either by biospecific elutions with different coenzymes or inhibitors, or by sequential passages of the extract through several cofactor-related affinity columns. Important principles were developed to purify enzymes exhibiting low affinity to the affinity columns. By “affinity filtration” of the extract through the affinity column, enzymes of low affinity can be retarded and separated effectively from strongly bound and nonadsorbed proteins. By the “saturation readsorption” procedure, enzymes of low affinity could be effectively separated from those of high affinity by overloading of the extracts on the affinity columns. Readsorption of the leaked low affinity enzymes to a second affinity column often results in better enzyme purification because of the elimination of competitive high affinity enzymes. With the application of these principles, the following enzymes and their genetic variants were highly purified via a single- or two-step affinity column procedure: lactate dehydrogenase-A, lactate dehydrogenase-B, lactate dehydrogenase-X, phosphoglycerate kinase-A, phosphoglycerate kinase-B, cytoplasmic and mitochondrial isocitrate dehydrogenase, malate dehydrogenase, malic enzyme, glucose-6-phosphate dehydrogenase, glutathione reductase, phosphoglucose isomerase and pyruvate kinase from mouse tissues; alcohol dehydrogenase, malate dehydrogenase, α-glycerol-phosphate dehydrogenase, malic enzyme, and glucose-6-phosphate dehydrogenase fromDrosophila. 相似文献
3.
The photosensitized oxidation of NADPH by oxygen can be used for the determination of the reduced coenzymes by means of a Clark oxygen electrode. This method is suitable for coupling to enzyme-catalysed dehydrogenation reactions and thus for the determination of glucose-6-phosphate with glucose-6-phosphate dehydrogenase and of glucose with the combined ATP/hexokinase/glucose-6-phosphate dehydrogenase system, even with the use of immobilized mediators. 相似文献
4.
Overexpression of glucose-6-phosphate dehydrogenase in genetically modified Saccharomyces cerevisiae
Lojudice Fernando H. Silva Daniel P. Zanchin Nilson I. T. Oliveira Carla C. Pessoa Adalberto 《Applied biochemistry and biotechnology》2001,91(1-9):161-169
Glucose-6-phosphate dehydrogenase (G6PD) (EC 1.1.1.49) is an abun dant enzyme in Saccharomyces cerevisiae. This enzyme is of great interest as an analytical reagent because it is used in a large number of quantitative assays. A
strain of S. cerevisiae was genetically modified to improve G6PD production during aerobic culture. The modifications are based on cloning the G6PD
sequence under the control of promoters that are upregulated by the carbon source used for yeast growth. The results showed
that S. cerevisiae acquired from a commercial source and the same strain produced by aerobic cultivation under controlled conditions provided
very similar G6PD. However, G6PD production by genetically modified S. cerevisiae produced very high enzyme activity and showed to be the most effective procedure to obtain glucose-6-phosphate dehydrogenase.
As a consequence, the cost of producing G6PD can be significantly reduced by using strains that contain levels of G6PD up
to 14-fold higher than the level of G6PD found in commercially available strains. 相似文献
5.
Phosphoglucomutase and glucose-6-phosphate dehydrogenase were immobilized to s-triazine trichloride activated cellulose. The optimal conditions for binding the immobilized enzymes were determined and the kinetic and physical properties were investigated.The final ratio of the two enzymes immobilized to the surface was determined by the physical properties of the enzymes as well as by the ratio of the enzymes present in the attachment solution. The immobilized enzymes were found to retain at least 60% of the original activity for at least 40 days when stored at 4°C and in the presence of substrates and cofactors. Immobilized phosphoglucomutase and glucose-6-phosphate dehydrogenase also were much more stable at 58°C, retaining 28 and 13% of the original activity, respectively, after 75 min. The apparentK m’s were 5.4 to 1.5 times higher for immobilized phosphoglucomutase and glucose-6-phosphate dehydrogenase, respectively than for the soluble enzymes. 相似文献
6.
《Analytical letters》2012,45(8):1861-1871
Abstract Flow-injection procedures for the indirect determination of glucose-6-phosphate dehydrogenase, glucose-6-phosphate, malate dehydrogenase and L-malate are described. They are based on a coupled reaction with bacterial luciferase and oxidoreductase co-immobilized on cyanogen bromide activated Sepharose 4B. the limits of detection are 0.015 pmol for glucose-6-phosphate dehydrogenase, 0.03 pmol for malate dehydrogenase, 10?8 mol?1 for glucose-6-phosphate and 10?6 mol l?1 for L-malate. the reproducibility is less than 5% relative standard deviation (n=5) for all assays and the sample throughput is 60 h?1. 相似文献
7.
Twelve enzymes from mature pollen grains of maize were separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The separation in the second dimension was both in the presence and absence of sodium dodecyl sulfate (SDS). Ten of the investigated enzymes lost activity after separation in the presence of SDS, but those of esterases and acid phosphatase could be recovered. On the other hand, 2-D electrophoresis without SDS is suitable for the analysis of maize pollen pectinesterase, malate dehydrogenase, glutamic-oxalacetic transaminase, diaphorase, superoxide dismutase, and phosphoglucose isomerase. 1-D PAGE and isoelectric focusing (IEF) are sufficient to analyze glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, shikimic dehydrogenase, and glutamate dehydrogenase. The possibility of applying 2-D electrophoresis for the analysis of enzymes from single stigma and stigma exudate is dicussed. 相似文献
8.
Glucose 6-phosphate dehydrogenase shows a high partition coefficient in poly-(ethylene glycol)-dextran aqueous two-phase systems in comparison with those for 6-phosphogluconate dehydrogenase, phosphofructokinase and the bulk of proteins present in rat erythrocyte haemolysates. As a consequence, fractions highly enriched in glucose 6-phosphate dehydrogenase can be obtained after multiple partitions in the above systems with a counter-current distribution procedure. Phosphofructokinase shows a high affinity for Cibacron Blue and, as a result, the enzyme can be extracted in the top phase of poly(ethylene glycol)-dextran systems containing Cibacron Blue-poly(ethylene glycol) (affinity systems). The efficiency for the purification of the enzymes by partitioning is increased up to 10-fold when enzyme-rich fractions, obtained by precipitation with poly(ethylene glycol), are used instead of original haemolysate. The recovery of enzyme activities is near 100% in both instances. 相似文献
9.
Lata S. Nerurkar Barbara J. Zeligs Joseph A. Bellanti 《Photochemistry and photobiology》1978,28(4-5):781-786
Abstract— The total superoxide dismutase activity of rabbit alveolar macrophages (AM) increased twofold during the first postnatal week. This increase in superoxide dismutase activity was primarily mitochondrial and paralleled the increase in the number of mitochondria in these cells which has been previously reported. The superoxide dismutase activity of AM in culture for 18 h was significantly increased by hyperoxia; catalase activity in hyperoxic conditions was slightly adversely affected; glucose-6-phosphate dehydrogenase activity was increased but not significantly over control values. Hyperoxic cultures beyond 42 h decreased the total number of viable cells and the superoxide dismutase activity expressed per viable cell; the disappearance of catalase and glucose-6-phosphate dehydrogenase activities in this period, however, was more rapid in control alveolar macrophages than those under hyperoxia. 相似文献
10.
This work presents the first known use of response surface methodology (RSM) in electrophoretically mediated microanalysis. This concept is demonstrated by examining the optimization of reaction conditions for the conversion of nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide, reduced form by glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) in the conversion of glucose-6-phosphate to 6-phosphogluconate. Experimental factors including voltage, enzyme concentration, and mixing time of reaction at the applied voltage were selected at three levels and tested in a Box-Behnken response surface design. Upon migration in a capillary under CE conditions, plugs of substrate and enzyme are injected separately in buffer and allowed to react at variable conditions. Extent of reaction and product ratios were subsequently determined by CE. The model predicted results are shown to be in good agreement (7.1% discrepancy difference) with experimental data. The use of chemometric RSM provides a direct relationship between electrophoretic conditions and product distribution of microscale reactions using CE, thereby offering a new and versatile approach to optimizing enzymatic experimental conditions. 相似文献
11.
Chemiluminometric methods are described for the automated flow injection analysis of NADPH and NADH using an immobilized enzyme column reactor and serum magnesium. This application is for the clinical analysis of NADPH and NADH. The reactor for NADPH and NADH contains immobilized L-glutamate dehydrogenase and L-glutamate oxidase, and that for serum magnesium immobilized hexokinase, glucose-6-phosphate dehydrogenase, L-glutamate dehydrogenase and L-glutamate oxidase. When the sample is introduced into the four-enzyme bioreactor, hydrogen peroxide is produced in proportion to the concentration of serum magnesium by the successive reactions. A co-immobilized hexokinase/glucose-6-phosphate dehydrogenase/glutamate dehydrogenase column reactor gave better efficiency compared with an enzyme column which was prepared by packing co-immobilized hexokinase/glucose-6-phosphate dehydrogenase and immobilized glutamate dehydrogenase to make two layers. Magnesium in serum was determined with 1 microL of the sample without carry-over and for an assay time of approximately 15 s. The present method is sensitive (detection limit 0.1 nmol) because Mg2+ is recycled in a column, and gives perfect linearity of the data up to 3.0 mmol/L with satisfactory precision, reproducibility, and accurate reaction recoveries. 相似文献
12.
Gerhard Michal Michael Nelböck Günther Weimann 《Analytical and bioanalytical chemistry》1970,252(2-3):183-188
The phosphorylase assay is performed in the direction from glycogen to glucose-1-phosphate by coupling with the glucose-6-phosphate dehydrogenase reaction. Using this test, some properties of the activation mechanisms in liver and muscle by cyclophosphates are compared and the results discussed. A large number of cyclophosphates is tested in both systems. Molecular variations in the cyclophosphates shift the activation curves equally in both systems. All cyclophosphates activate the system to an equal degree. 相似文献
13.
G. F. Deitzer D. W. Hopkins U. Haertlé E. Wagner 《Photochemistry and photobiology》1978,27(2):127-131
An approach to determine the photomorphogenic effect of light (white or continuous far-red) on the development of rhythmic enzyme activity in Chenopodium rubrum L. is described. Previous results, obtained from mature seedlings grown in white light, demonstrated stable oscillations with periods ranging between 12 and 15 h for all of the enzymes tested. The present results, obtained during deetiolation, were complicated by the presence of a higher frequency component with a period of about 6 h. When the various oscillating components were defined, the analysis showed: (1) the enzymes of the Krebs cycle (malate and isocitrate dehydrogenase), the closely associated glutamate dehydrogen-ase, and the glycolytic pathway ((NAD) glyceraldehyde-3-phosphate dehydrogenase) had a dominant period in the range of 12–15 h, (2) those of the oxidative pentose phosphate pathway were either weakly circadian (glucose-6-phosphate dehydrogenase) or apparently arhythmic (6-phosphogluconate dehydrogenase), (3) the (NADP) glyceraldehyde-3-phosphate dehydrogenase from the Calvin cycle was circadian when kept in continuous darkness but becomes 15 h when placed in light, and (4) only the Calvin cycle enzyme is affected by light in the level of its activity and in its oscillatory behavior. 相似文献
14.
《Analytical letters》2012,45(21-22):2155-2167
Abstract A bienzyme electrode for glucose-6-phosphate and ATP based on the sequential reactions of coimmobilized glucose-6-phosphate dehydrogenase and hexokinase and for fructose using the competition with glucose has been developed. Electrochemical indication is performed using air-oxidation of reduced N-methylphenazinium ion. The sensor responds linearly to fructose up to 3 mM with a lower detection limit of 0.3 mM. 相似文献
15.
Necmettin Yildirim Mehmet Çiftçi Ö. İrfan Küfrevioğlu 《Journal of mathematical chemistry》2002,31(1):121-130
This paper describes a general methodology to handle closed multi enzyme systems using mixture of symbolic (which depends on the Gröbner Basis technique) and numerical computation methods. The applicability of the proposed method has been examined for the closed three-enzyme system of rabbit heart creatine kinase (EC 2.7.3.2), yeast hexokinase (EC 2.7.1.1) and human erythrocyte glucose 6-phosphate dehydrogenase (EC 1.1.1.49) using experimental data. 相似文献
16.
A Kamei 《Chemical & pharmaceutical bulletin》1992,40(10):2787-2791
To learn whether glycation plays a role in insolubilization or in senile cataractogenesis, the reactivity of lens protein from normal and senile cataractous lenses and individual crystallin prepared from human lens with various sugars [glucose, glucose-1-phosphate (G-1-P), glucose-6-phosphate (G-6-P) and fructose], and the insolubility of those proteins were determined. The reactivity of human lens protein to glucose was increased in a dose-dependent manner, and it was demonstrated that 17.9, 18.5 and 24 kDa proteins were susceptible to glycation with sugars. The study also showed that alpha-, beta-crystallins and high molecular weight (HMW) aggregate obtained from cataractous lens have some weak reactivity against sugars. It was demonstrated that the proteins obtained from normal lens of older age and from cataractous lenses have higher insolubilities to glucose than do normal younger ones. Measurement of glycosylated protein by affinity column chromatography revealed that cataractous lenses contained a larger amount of glycosylated protein than normal ones. These results suggest that there is an age-related increase of glycation in normal human lens protein, and that such glycation increases the amount of insolubilized protein with the effect of aging. The author also speculates that an abnormal acceleration of glycation in the human lens may induce senile cataract formation. 相似文献
17.
Christian R. Gomez Ingrid Espinoza Fazlay S. Faruque Md. Mahbub Hasan Khondaker Miraz Rahman Larry A. Walker Ilias Muhammad 《Molecules (Basel, Switzerland)》2021,26(4)
To date very few promising leads from natural products (NP) secondary metabolites with antiviral and immunomodulatory properties have been identified for promising/potential intervention for COVID-19. Using in-silico docking studies and genome based various molecular targets, and their in vitro anti-SARS CoV-2 activities against whole cell and/or selected protein targets, we select a few compounds of interest, which can be used as potential leads to counteract effects of uncontrolled innate immune responses, in particular those related to the cytokine storm. A critical factor for prevention and treatment of SARS-CoV-2 infection relates to factors independent of viral infection or host response. They include population-related variables such as concurrent comorbidities and genetic factors critically relevant to COVID-19 health disparities. We discuss population risk factors related to SARS-CoV-2. In addition, we focus on virulence related to glucose-6-phosphate dehydrogenase deficiency (G6PDd), the most common human enzymopathy. Review of data on the response of individuals and communities with high prevalence of G6PDd to NP, prompts us to propose the rationale for a population-specific management approach to rationalize design of therapeutic interventions of SARS-CoV-2 infection, based on use of NP. This strategy may lead to personalized approaches and improve disease-related outcomes. 相似文献
18.
Two fully enzymatic methods, colorimetric and fluorimetric, are reported for the determination of creatine kinase (EC 2.7.3.2) MB isoenzyme after immune-inhibition with the use of goat anti-human CK-M IgG antibodies. The residual creatine kinase activity is assayed by using hexokinase and glucose-6-phosphate dehydrogenase systems; the resulting NADPH is determined spectrophotometrically by reaction with p-iodonitro-tetrazolium violet in the presence of diaphorase as an intermediate electron carrier, or fluorimetrically by coupling the NADPH with resazurin/diaphorase to form resorufin. Both assays take only 12 min and require only 100 or 25μl of serum. The calibration plots of enzyme activities are linear up to 580 and 435 U l-1 of CK-MB in serum for the spectrophotometric and fluorimetric assays, respectively, the coefficients of variation being 2.3% and 4.6%, and the recovery values 103% and 100% respectively. The results correlate very well with those obtained by the Helena electrophoresis—fluoridensitometric methods. As little as 4 U l-1 and 2 U l-1 CK-MB could be measured reproducibly. 相似文献
19.
Human erythrocyte transketolase (sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehyde-transferase) was purified 8200-fold by adsorption onto hydroxylapatite, DEAE-cellulose treatment, acetone fractionation, and chromatography on Sephadex G-100. The purified transketolase could not be separated from glyceraldehyde-3-phosphate dehydrogenase, whereas the latter enzyme could be isolated in a pure state. Its homogeneity is suggested by sedimentation velocity, sedimentation equilibrium, and acrylamide electrophoresis. A molecular weight of 136000 was found. The physicochemical properties of glyceraldehyde-3-phosphate dehydrogenase and transketolase are very similar. A molecular weight of 136000 is suggested for transketolase, although gel filtration with Sephadex G-100 gave only 104000 ± 10%. This discrepancy is a reflection of an interaction of transketolase with the gel filtration medium. The isoelectric point for transketolase as well as for glyceraldehyde-3-phosphate dehydrogenase, as determined by isoelectric focussing, was found to be around 8.5. The activity of the enzyme is close to the maximum for pH 7.5 to pH 8.6. Additions of thiamine pyrophosphate or other cofactors do not influence the activity. Several divalent cations were tested. Sulfate and phosphate inhibit transketolase approximately to 50% between 50 and 100 mM concentration. Thiamine was present in transketolase, as shown by a microbiological assay and by the thiochrome reaction. The activation energy for the formation of sedoheptulose-7-phosphate from xylulose-5-phosphate was estimated from rate measurements to be 11.2 kcal/mole in the temperature range from 5° to 55°. 相似文献
20.
Protein partitioning in poly(ethylene glycol)/sodium polyacrylate aqueous two-phase systems 总被引:3,自引:0,他引:3
The partition of hemoglobin, lysozyme and glucose-6-phosphate dehydrogenase (G6PDH) in a novel inexpensive aqueous two-phase system (ATPS) composed by poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA) has been studied. The effect of NaCl and Na(2)SO(4), pH and PEG molecular size on the partitioning has been studied. At high pH (above 9), hemoglobin partitions strongly to the PEG-phase. Although some precipitation of hemoglobin occurs, high recovery values are obtained particularly for lysozyme and G6PDH. The partitioning forces are dominated by the hydrophobic and electrochemical (salt) effects, since the positively charged lysozyme and negatively charged G6PDH partitions to the non-charged PEG and the strongly negatively charged polyacrylate enriched phase, respectively. 相似文献